Relative fluorescence of normal and acid lipase-deficient cultured fibroblasts following administration of pyrene decanoic acid.

Skin fibroblasts, derived from normal individuals or patients with Wolman's disease (an autosomal recessive disorder due to acid lysosomal lipase deficiency) were incubated with the fluorescent fatty acid, pyrene-decanoic acid (P10). Measurements of the fluorescence intensities of the total lipid extracts indicated that equal quantities of P10 were incorporated into both cell types. The fluorescence emitted by the intact cells was subsequently recorded in a fluorescence microscope equipped with a microdetector unit, which permitted determination of the fluorescence emitted by the intact cell or by specific regions thereof. The fluorescence intensities emitted by the lipidotic cells exceeded those of their normal counterparts 2- and 5-fold when comparing the entire cells or the perinuclear region, respectively. The cells were then subjected to subcellular fractionation and an analysis of the fractions revealed that up to 85-90% of the fluorescence of the lysosome-mitochondrial pellet was derived from free pyrenedecanoic acid; the latter contributed only 15-18% to the fluorescence of the homogenate or the cytosol. There was no difference in the fluorescence of the lipid extracts from the respective fractions of the lipidotic or normal cells. However, the fluorescence emitted by the intact lysosome-mitochondrial fraction of the lipidotic cells exceeded that of its normal counterpart 2.5-fold. These data suggest that the increased fluorescence intensity of the intact lipidotic cells resulted from a higher quantum yield of free P10 molecules solubilized in the hydrophobic environment of their neutral lipid-containing storage granules.